Late stage results from the probe development effort to identify activators of signal transducer and activator of transcription 3 (STAT3).
Name: Late stage results from the probe development effort to identify activators of signal transducer and activator of transcription 3 (STAT3). ..more
BioActive Compounds: 8
Source (MLSCN Center Name): The Scripps Research Institute Molecular Screening Center
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: David Frank, Dana Farber Cancer Institute
Network: Molecular Library Probe Production Center Network (MLPCN)
Grant Proposal Number: 1 X01 MH079826-01
Grant Proposal PI: David Frank, Dana Farber Cancer Institute
External Assay ID: STAT3_ACT_PROBES_LATE STAGE
Name: Late stage results from the probe development effort to identify activators of signal transducer and activator of transcription 3 (STAT3).
The signal transducer and activator of transcription (STAT) family of transcription factors transduce signals from a variety of extracellular stimuli and are important mediators of inflammation, cell survival, differentiation, and proliferation (1, 2). STATs are activated in response to growth factors, cytokines, and G-CSF binding to cell surface receptor tyrosine kinases (1-3). In resting cells STATs are inactive in the cytoplasm. In response to stimuli, STATs are phosphorylated by the Janus-activated kinases (Jaks), which induces STAT dimerization and nuclear translocation, where STATs bind to specific enhancer elements in target genes (2). Although structurally similar, the seven STAT family member (STATs 1, 2, 3, 4, 5a, 5b, and 6) possess diverse biological roles (2). For example, STAT1 activation is pro-inflammatory and anti-proliferative, while STAT3 activation is anti-inflammatory and pro-apoptotic (2). STAT1 is largely responsible for mediating the effects of IFN-??, while STAT3 is predominantly involved in IL-6 signaling (4). STAT1 induces expression of genes that inhibit the cell cycle, and thus STAT1 is considered to have tumor suppressor properties (5). Studies show that STAT3 is activated in a majority of breast and prostate cancers, and that STAT3 inhibition using RNA interference or a dominant negative leads to reduced cell proliferation, survival, and wound healing (1, 4, 6). Blocking STAT3 interaction with the epidermal growth factor receptor (EGFR) using peptide aptamers has been shown to reduce tumor growth (7). Due to the diverse roles and potent phenotypes associated with STAT signaling, the identification of selective modulators of STAT3 activity may lead to pharmacological tools for cancer, wound healing, and inflammatory diseases.
Summary of Probe Development Effort:
Following primary HTS in singlicate to identify STAT3 activators (AID 871), primary screening in singlicate to identify STAT1 activators (AID 932), confirmation of STAT3 hit activity in triplicate (AID 1267), counterscreening in triplicate against NFkB (AID 1309) and STAT1 (AID 1318) to determine selectivity, followed by titration assays to determine compound potency (AID 1398) and selectivity (AID 1406), compounds (SID 14735210) were identified as candidates for probe development. The probe candidate was purchased in powder form (SID 87326010), along with analogs belonging to the isoxazole carboxamide scaffold, which were either purchased in powder form, re-ordered from the MLSMR in liquid form, or synthesized the SRIMSC. These compounds were tested in dose response assays against STAT3 and STAT1, as well as additional counterscreening assays to determine cytotoxicity, and qPCR assays by the assay provider. The probe candidate (SID14735210/SID 87326010) confirmed activity and is claimed as a potent, selective, and non-toxic STAT3 activator probe.
As prescribed in the CPDP, the chief goal for this probe development project was to find a selective activator the signal transducer and activator of transcription 3 (STAT3). In response to this goal, the probe compound (SID 14735210 liquid/ SID 87326010 powder), belonging to the isoxazole carboxamide scaffold, is claimed as a potent and selective activator of STAT3. This probe has a low nanomolar EC50 for STAT3 and is inactive against the related STAT1 and NF?B anti-targets. As expected for a useful STAT3 activator, QPCR experiments (run by the assay provider) confirm that the probe upregulates transcription of the oncogene BCL3. In addition, the probe does not exhibit cytotoxicity in the parental cell lines used for the STAT3 assay (HT-1080) and STAT1 assay (NIH-3T3).
In contrast, prior art Compound 1 is a non-selective xanthocillin reported to induce phosphorylation of STAT3, Jak2, and STAT5 in a human leukemia cell line (9). As a potent EGFR tyrosine kinase inhibitor, prior art Compound 56 demonstrated potency in the STAT3 activator assay; however, it was equally potent in the STAT1 activator assay.
The probe was identified from the STAT3 uHTS campaign. It was selected as a starting point for medicinal chemistry because it increased expression of BCL3, a known STAT3-dependent oncogene. It was selected as a probe since analogs that were generated during the probe development effort were either less potent against STAT3 (analogs 1, 3 and 4), or did not significantly increase BCL3 transcription (e.g., analog 2).
Recommendations for the scientific use of this probe:
Taken together, the results of the screening campaign, assay provider studies, and various probe development assays described herein validate the selection of SID 14735210 liquid (SID 87326010 powder) as a potent and selective STAT3 activator probe with cell-based activity. In the light that the prior art STAT3 activators have off-target activity, the probe identified here is useful for oncologic research aiming to elucidate the STAT3 signaling pathway, with minimal effect on related STAT1 and NF?B pathways.
The above probe development effort resulted in the identification of one probe. A probe report has been published (http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html). Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs below. Please also see Summary AID 1805.
1. Alvarez JV, Febbo PG, Ramaswamy S, Loda M, Richardson A, Frank DA. Identification of a genetic signature of activated signal transducer and activator of transcription 3 in human tumors. Cancer Res. 2005 Jun 15;65(12):5054-62.
2. Schindler C, Levy DE, Decker T. JAK-STAT signaling: from interferons to cytokines. J Biol Chem. 2007 Jul 13;282(28):20059-63.
3. Germain D, Frank DA. Targeting the cytoplasmic and nuclear functions of signal transducers and activators of transcription 3 for cancer therapy. Clin Cancer Res. 2007 Oct 1;13(19):5665-9.
4. Levy DE, Darnell JE Jr. Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol. 2002 Sep;3(9):651-62.
5. Battle TE, Wierda WG, Rassenti LZ, Zahrieh D, Neuberg D, Kipps TJ, Frank DA. In vivo activation of signal transducer and activator of transcription 1 after CD154 gene therapy for chronic lymphocytic leukemia is associated with clinical and immunologic response. Clin Cancer Res. 2003 Jun;9(6):2166-72.
6. Takeda, K. Takeda K, Kaisho T, Yoshida N, Takeda J, Kishimoto T, Akira S.1998. Stat3 activation is responsible for IL-6-dependent T cell proliferation through preventing apoptosis: generation and characterization of T cell- specific Stat3-deficient mice. J. Immunol. 161:4652-4660.
7. Buerger C, Nagel-Wolfrum K, Kunz C, Wittig I, Butz K, Hoppe-Seyler F, Groner B. Sequence-specific peptide aptamers, interacting with the intracellular domain of the epidermal growth factor receptor, interfere with Stat3 activation and inhibit the growth of tumor cells. J Biol Chem. 2003 Sep 26;278(39):37610-21.
8. Nelson EA, Walker SR, Kepich A, Gashin LB, Hideshima T, Ikeda H, Chauhan D, Anderson KC, Frank DA. Nifuroxazide inhibits survival of multiple myeloma cells by directly inhibiting STAT3. Blood. 2008 Dec 15;112(13):5095-102.
9. Sakai R, Nakamura T, Nishino T, Yamamoto M, Miyamura A, Miyamoto H, Ishiwata N, Komatsu N, Kamiya H, Tsuruzoe N. Xanthocillins as thrombopoietin mimetic small molecules. Bioorg Med Chem. 2005 Dec 1;13(23):6388-93.
Late stage, probes, probe, STAT3, signal transducer and activator of transcription 3, acute-phase response factor, APRF, activation, activator, potentiation, potentiator, U3A, transcription factor, luciferase, luminescence, reporter, dose response, counterscreen, 1536, HTS, assay, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
§ Panel component ID.
Please see AIDs 871, 932, 1267, 1309, 1318, 1398, 1406, Summary AID 1805, and below for protocols performed in this probe development effort. Assays with the tested powder samples are described below.
STAT3 Activation Assay (Assay 1):
The purpose of these assays is to identify compounds that increase STAT3 activity. Activation of STAT3 transcription was measured using a human U3A fibrosarcoma cell line that stably expresses a human STAT3::luciferase construct. This cell line is deficient in STAT1. In this assay, test compounds were screened for their ability to increase IL-6-mediated STAT3::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with IL-6 to activate STAT3 transcription. Changes in STAT3::luciferase activity were monitored by measuring luminescence. As designed, a STAT3 activator will enhance IL-6-mediated STAT3 transcription, thus increasing the activation of the luciferase reporter gene, and increasing luminescence. Compounds were tested in a 10-point, 1:3 dilution series in triplicate, starting at a nominal test concentration of 55.7 micromolar.
STAT1 Activation Counterscreen Assay (Assay 2):
The purpose of these assays is to identify compounds that increase STAT1 activity. These assays also serves as counterscreen to determine whether compounds identified as active in the STAT3 activation assays were nonselective due to activation of STAT1. Activation of STAT1 activity was measured using a murine NIH 3T3 fibroblast cell line cell line that stably expresses a STAT1::luciferase construct. In these assays, test compounds were screened for their ability to increase IFN-gamma-mediated STAT1::luciferase reporter activity. Cells were exposed to test compounds, followed by treatment with IFN-gamma. Changes in STAT1::luciferase activity were monitored by measuring luminescence. As designed, a STAT1 activator will enhance IFN-gamma-mediated STAT1 transcription, thus increasing the activation of the luciferase reporter gene, and increasing luminescence. Compounds were tested in a 10-point, 1:3 dilution series in triplicate, starting at a nominal test concentration of 55.7 micromolar.
Cytotoxicity Counterscreen Assay (Assay 3):
The purpose of this assay is to determine the cytotoxicity of compounds identified as probe candidates. This assay employs the CellTiter-Glo luminescent reagent, which contains luciferase to catalyze the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. As designed, cytotoxic compounds will reduce viable cell numbers and ATP levels, resulting in decreased well luminescence. Compounds were assayed in a 10-point 1:3 dilution series starting at a nominal concentration of 56 micromolar.
BCL3 QPCR Assay (Assay 4):
The purpose of this assay is to determine whether compounds of interest can activate expression of the STAT3 target gene, BCL3. The assay protocol has been described (8). Briefly, cells were incubated with either compound or vehicle, and RNA was isolated using the RNeasy kit (QIAGEN, Valencia, CA). cDNA was generated using the Taqman reverse transcription kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicate using SYBR green master mix (Applied Biosystems) on a model 7500 real time PCR system (Applied Biosystems). Data are expressed as the mean fold change plus or minus SE of 3 replicates. Each assay was repeated at least 3 times.
Probes were identified. Not all compounds were tested in all biological assays, as indicated.
The following compounds were not tested in the Cytotoxicity and qPCR assays due to lower STAT3 target activity and/or nonselectivity, compared to the probe SID14735210 liquid/ SID87326010 powder: SIDs 57287818, 57288067, 57288064, 57288070, 57288068, 57288069, 57288063, 85261423, 85261424, 85261425, 85261426.
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* Activity Concentration. ** Test Concentration. § Panel component ID.